CN110257339A - The cell line and its construction method of expression anti-new castle disease virus fusion protein and application - Google Patents
The cell line and its construction method of expression anti-new castle disease virus fusion protein and application Download PDFInfo
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- CN110257339A CN110257339A CN201910543875.9A CN201910543875A CN110257339A CN 110257339 A CN110257339 A CN 110257339A CN 201910543875 A CN201910543875 A CN 201910543875A CN 110257339 A CN110257339 A CN 110257339A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The invention discloses the cell line of expression anti-new castle disease virus NP protein nano antibody-HRP fusion protein, the cell line is C201981 in the deposit number of China typical culture collection center；A kind of construction method of cell line is also provided, successfully the slow virus carrier for carrying nano antibody-HRP fusion can be transferred in the cell strain for expressing the foreign gene by the construction method, and stablize expression nano antibody-HRP fusion protein；Also disclose the cell line of thus construction method building, and application of the fusion protein of cell line expression in serum detection, it is applied particularly to the detection of newcastle epidemic disease antibody in chicken serum, supernatant after cell line culture through the invention, by the combination situation for directly detecting positive chicken serum newcastle epidemic disease antibody and Newcastle disease virus NP albumen after potency 1:1000 dilution, judge whether to infect newcastle disease virus, this detection method is simple and easy to operate, production cost is reduced, and improves detection efficiency.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of cell line for expressing newcastle disease virus fusion protein and its
Construction method and application, and in particular to a kind of cell for expressing anti-new castle disease virus NP protein nano antibody-HRP fusion protein
System and its construction method and application.
Background technique
Newcastle disease (Newcastle disease, ND) be by newcastle disease virus (Newcastle disease virus,
NDV a kind of acute, highly contagious disease caused by) based on infected poultry.The disease is with alimentary canal and respiratory disease
For main feature, morbidity and mortality are high, bring serious harm to aviculture.Newcastle disease virus gene group encodes 6 kinds
Structural proteins, respectively memebrane protein, phosphorylated protein, nucleocapsid protein (NP), hemagglutinin-neuraminidase albumen, fusion egg
White and high molecular weight protein (L).NP albumen is made of 489 amino acid residues, and molecular weight is about 53kDa, which is NDV core clothing
The major protein components of shell are the most abundant albumen of content in virion, with natural immunogenicity, but do not have
There is immanoprotection action, therefore is commonly used as the clinical diagnosis of monitoring vaccination program and newcastle disease.
Currently, the main virulent separation of NDV detection method and identification, Electronic Speculum detection technique, serological diagnostic method with
And molecular biological testing etc..ELISA method is a kind of current most widely used immunologic detection method, has sensibility
Good, high specificity, it is easy to operate, reproducible the advantages that.The ELISA kit for clinically detecting chicken source NDV antibody is wide
General production and use, but in such kit production procedure or need to prepare the secondary antibody for the anti-chicken that enzyme marks (indirectly
ELISA), or the monoclonal antibody of anti-NDV nucleocapsid protein is needed, therefore, has that production process is complicated, at high cost, commercial reagents
The disadvantages of ingredient that the assembling of box needs is more, such as: the highest detection chicken from IDEXX company of existing market frequency of use
The ELSA kit of anti-NDV antibody in serum, the kit need to prepare the goat-anti chicken of horseradish peroxidase (HRP) label
Secondary antibody, production routine, then by its immune sheep, after separating goat-anti chicken IgY, then are carried out firstly the need of chicken IgY is isolated and purified
The label of enzyme, generating process is complicated, causes at high cost.
Nano antibody is a kind of natural deletions light chain that Hamers-Casterman in 1989 etc. has found in camel blood
Heavy chain antibody, molecular weight 15kDa, diameter 2.2nm, long 4.8nm, compared with conventional antibodies, nano antibody have it is higher
Affinity and water-soluble, conformational stability are easy to genetic engineering transformation and pass through the advantages such as blood-brain barrier.In recent years, with right
Nano antibody research deepens continuously, which visualizes tracer, structure elucidation and human and animal's epidemic disease in albumen
It is used widely in diagnosis and treatment field etc..Therefore, using nano antibody advantage, anti-NDV core clothing will be encoded by finding one kind and capable of stablizing
The cell line of the nano antibody gene expression of glutelin is applied directly to detection blood by the nano antibody that cell line stablizes expression
In the ELISE technology of clear NDV antibody, it will simplify the cumbersome enzyme of existing ELISA method marking program, so that producing
Journey is simple and reduces production cost.
Summary of the invention
It is an object of the invention to solve at least the above problems, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide a kind of expression anti-new castle disease virus NP protein nano antibody to merge egg with HRP
White cell line, the cell line have the energy of the nano antibody and HRP fusion protein of stablizing expression anti-new castle disease virus NP albumen
Power.
It is a still further object of the present invention to provide a kind of expression anti-new castle disease virus NP protein nano antibody to merge egg with HRP
The construction method of white cell line, the process for being constructed and being packed by slow virus carrier successfully obtain to stablize and express anti-newcastle disease
The cell line of viral NP protein nano antibody and HRP fusion protein.
It is a still further object of the present invention to provide a kind of anti-new castle disease virus NP protein nano antibody and HRP fusion protein to exist
Application in newcastle epidemic disease antibody detection, the fusion protein can be anti-with positive serum in the positive serum of anti-new castle disease virus
The antigen binding with newcastle disease virus nucleocapsid protein of body competition, can simplify the detection method of newcastle disease virus.
It is a still further object of the present invention to provide a kind of anti-new castle disease virus NP protein nano antibody and HRP fusion protein to exist
Application in the detection of anti-new castle disease virus positive chicken serum, cell line culture supernatant of the present invention is added in positive chicken serum, can
The combination of the fusion protein and Newcastle disease virus NP albumen is blocked, conducive to the method for simplifying chicken infection anti-new castle disease virus detection.
In order to realize object of the present invention and further advantage, provides a kind of expression anti-new castle disease virus NP albumen and receive
The cell line of meter Kang Ti-HRP fusion protein, the cell line are in the deposit number of China typical culture collection center
C201397；Depositary institution address: Hubei Wuhan, Wuhan University.
The present invention provides the cell that anti-new castle disease virus NP protein nano antibody-HRP fusion protein is expressed described in one kind
The construction method of system, which comprises the following steps:
A: Newcastle disease virus NP will composite coding anti-new castle disease virus NP protein nano antibody and HRP fusion: be encoded
The nucleotide sequence of protein nano antibody and HRP directly merge, and the flexible sequence connection in centre synthesizes recombinant fusion gene；
B: the recombined lentivirus vector for expressing the recombinant fusion gene is constructed: by the recombinant fusion gene through enzyme
It after cutting, is connected on slow virus carrier, obtains recombined lentivirus vector；Wherein, slow virus carrier PLVX-IRES-
ZsGreen1；
C: the packaging of recombined lentivirus vector: the recombined lentivirus vector and 2 helper plasmids are transfected to culture
In HEK-293T cell, secondary culture, screening obtains expression anti-new castle disease virus NP protein nano antibody and HRP fusion protein
Cell line.
Preferably, the nucleotide sequence of the Newcastle disease virus NP protein nano antibody is as shown in SEQ ID NO:1, institute
The nucleotide sequence of HRP is stated as shown in SEQ ID NO:2.
Preferably, the helper plasmid is psPAXA2 and pMD2.G.
The present invention also provides a kind of cell lines for expressing anti-new castle disease virus NP protein nano antibody-HRP fusion protein to be
It is constructed by the construction method.
The present invention also provides a kind of anti-new castle disease virus NP protein nano antibody-HRP fusion proteins by the cell line point
Secrete expression.
The present invention also provides the anti-new castle disease virus NP protein nano antibody-HRP fusion proteins described in one kind in newcastle disease
Application in antibody test.
The present invention also provides the anti-new castle disease virus NP protein nano antibody-HRP fusion proteins described in one kind in anti-new city
Application in the detection of epidemic disease virus-positive chicken serum.
The present invention is include at least the following beneficial effects:
The cell line of expression anti-new castle disease virus NP protein nano antibody-HRP fusion protein of the present invention, training
Contain a large amount of anti-new castle disease virus NP protein nano antibody-HRP fusion proteins in feeding supernatant；By expressing anti-newcastle disease
The construction method of the cell line of viral NP protein nano antibody-HRP fusion protein, will have anti-new castle disease virus NP albumen to receive
For the recombination Successful transfection of meter Kang Ti and HRP fusion protein to HEK-293T cell, composition, which can be stablized, expresses the fusion protein
Cell line；The anti-new castle disease virus NP protein nano antibody-HRP fusion protein that the cell line is expressed is applied to serum inspection
In survey, it can avoid indirect ELISA in the prior art or need the monoclonal antibody of anti-NDV nucleocapsid protein in detection newcastle epidemic disease antibody side
Complicated procedures and ELIAS secondary antibody in method increase production cost, reduce detection efficiency；And the fusion for specifically expressing the cell line
Albumen is applied in the detection of positive chicken serum, and the supernatant after chicken serum to be detected and the cell line culture is diluted 1000 times
Liquid mixed culture, can block reacting for nanoprotein-HRP fusion protein and anti-new castle disease virus NP albumen, therefore, structure of the present invention
The cell line built is through cultivating the anti-new castle disease virus nanoprotein-HRP fusion protein of secreting, expressing, it is only necessary to be trained with the cell line
Feeding supernatant can be used to whether chicken infects in the detection method of newcastle disease virus, avoid anti-new city in existing detection chicken serum
The cumbersome process such as ELIAS secondary antibody, greatly improves detection efficiency in the ELSA kit of epidemic disease antiviral antibody, moreover it is possible to reduce detection at
This.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is the digestion qualification result after synthesis NDV-Nb5-HRP fusion of the present invention；
Fig. 2 is the HEK-293 cell line for stablizing expression NDV-NP-Nb5-HRP of the screening under fluorescence microscope
Observed result；
Fig. 3 is the activity of HRP in the supernatant for the cell line that the Salmonella detection is surely sieved；
Fig. 4 is that the indirect ELISA detects the NDV-NP-Nb5-HRP of steady sieve cell system secretion and the specificity of antigen
In conjunction with level；
Fig. 5 is NDV- in the cell pyrolysis liquid and culture supernatant of the Western blotting detection screening cell
The result of NP-Nb5-HRP expression quantity；
Fig. 6 a is the amplification of the LaSota plants of NP genes of anti-new castle disease virus；
Fig. 6 b is the digestion qualification result of the recombinant prokaryotic expression vector of the building；
Fig. 7 a is the expression of results that the SDS-PAGE analyzes nucleocapsid protein；
Fig. 7 b is antigenic result that the Western blot analyzes prokaryotic expression protein；
Fig. 8 is the combination water of NDV-NP-Nb5-HRP and NDV-NP albumen in the newcastle disease virus antibody chicken serum
It is flat；A is the testing result of the hole 1-48 serum, and b is the testing result of the hole 49-96 serum；
Fig. 9 is the map of the slow virus carrier pLVX-IRES-ZsGreen1；
Figure 10 is the map of the helper plasmid psPAX2；
Figure 11 is the map of the helper plasmid pMD2.G.
Specific embodiment
The present invention is described in further detail below, to enable those skilled in the art's refer to the instruction text being capable of evidence
To implement.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded one or more
The presence or addition of a other elements or combinations thereof.
The present invention provides a kind of cell line for expressing anti-new castle disease virus NP protein nano antibody-HRP fusion protein, life
Entitled human embryo kidney (HEK) transformed cells HEK293T-NDV-Nb5-HRP, guarantor of the cell line in China typical culture collection center
Hiding number is C201981；Depositary institution address: Hubei Wuhan, Wuhan University, preservation time are 2019.05.29.
The present invention also provides the thin of the expression anti-new castle disease virus NP protein nano antibody-HRP fusion protein described in one kind
The construction method of born of the same parents system, comprising the following steps:
A: Newcastle disease virus NP will composite coding anti-new castle disease virus NP protein nano antibody and HRP fusion: be encoded
The nucleotide sequence of protein nano antibody and HRP directly merge, and the flexible sequence connection in centre synthesizes recombinant fusion gene；
B: the recombined lentivirus vector for expressing the recombinant fusion gene is constructed: by the recombinant fusion gene through enzyme
It after cutting, is connected on slow virus carrier, obtains recombined lentivirus vector；Wherein, slow virus carrier pLVX-IRES-
ZsGreen1；
C: the packaging of recombined lentivirus vector: the recombined lentivirus vector and 2 helper plasmids are transfected to culture
In HEK-293T cell, secondary culture, screening obtains expression anti-new castle disease virus NP protein nano antibody and HRP fusion protein
Cell line.
In one preferred embodiment, the nucleotide sequence of the Newcastle disease virus NP protein nano antibody such as SEQ ID NO:1
Shown, the nucleotide sequence of the HRP is as shown in SEQ ID NO:1.
In one preferred embodiment, the helper plasmid is psPAX2 and pMD2.G.
The present invention also provides a kind of cell lines for expressing anti-new castle disease virus NP protein nano antibody-HRP fusion protein to be
It is constructed by the construction method.
The present invention also provides a kind of anti-new castle disease virus NP protein nano antibody-HRP fusion proteins by the cell line point
Secrete expression.
The present invention also provides the anti-new castle disease virus NP protein nano antibody-HRP fusion proteins described in one kind in newcastle disease
Application in antibody survey.
The present invention also provides the anti-new castle disease virus NP protein nano antibody-HRP fusion proteins described in one kind in anti-new city
Application in the detection of epidemic disease virus-positive chicken serum.
Anti-new castle disease virus NP protein nano antibody provided by the invention, specially anti-new castle disease virus LaSota plants of NP eggs
White nano antibody, is named as Nb5, is made of 122 amino acid, amino acid sequence is as shown in SEQ NO.3；Horseradish peroxide
Compound enzyme, i.e. HRP are made of 310 amino acid, and amino acid sequence is as shown in SEQ NO.4.
Embodiment 1
1, the building of the cell line of anti-new castle disease virus NP protein nano antibody-HRP fusion protein is expressed
The building and packaging of 1.1 recombined lentivirus vectors
1.1.1 the gene order of composite coding anti-new castle disease virus NP albumen Nb5-HRP fusion protein
Nucleotide sequence according to coding anti-new castle disease virus NP protein nano antibody is as shown in SEQ ID NO:1 and HRP
Nucleotide sequence as shown in SEQ ID NO:2, by Suzhou, Jin Weizhi biotech company is directly synthesized, the flexible sequence in centre
(AGTAGTAGTGGCAGTGGT) it is attached, and His label is added in the c-terminus of fusion
(CATCACCACCATCAC), it is easy to subsequent detection；EcoR I and BamH I is added at the 5 ' of recombinant fusion gene and 3 ' ends simultaneously
Restriction enzyme site synthesizes gene NDV-Nb5-HRP and is connected into pUC57 carrier, digestion removes EcoR I convenient for the building of recombinant vector
It is identified after restriction enzyme site GAATTC and BamH I restriction enzyme site GGATCC.
Fig. 1 is the digestion qualification result synthesized after NDV-Nb5-HRP fusion, the results show that obtaining after digestion
1123bp segment, it was demonstrated that successfully synthesize anti-new castle disease virus NP protein nano antibody-HRP fusion.
1.1.2 the building of recombined lentivirus vector
By the recombinant fusion gene product of 1.1.1 synthesis and slow virus carrier pLVX-IRES-ZsGreen1 (such as Fig. 9 institute
Show, be purchased from Clontech company) double digestion is carried out with EcoR I and BamH I simultaneously, T4 ligase is then utilized, by digestion
Recombinant fusion gene product is connected in slow virus carrier pLVX-IRES-ZsGreen1, conversion to DH5 α competent cell
(TransGen Biotech) carries out the conversion of recombinant plasmid according to its operating instruction.Utilize plasmid extraction kit
(TransGen Biotech) extracts plasmid by kit operating instruction and carries out digestion identification and sequencing.
Correct recombined lentivirus vector carrier is sequenced and is named as PLVX-NDV-Nb5-HRP-IRES-ZsGreen1.
1.1.3 the packaging of recombined lentivirus vector
HEK-293T cell is cultivated using the DMEM culture medium containing 10% calf serum, and HEK-293T cell is spread
To 6 orifice plates, 1 × 105A cells/well reaches 70% or so to cell confluency degree, by the recombined lentivirus vector PLVX- of building
NDV-Nb5-HRP-IRES-ZsGreen1, helper plasmid psPAX2 (map is as shown in Figure 10, be purchased from Clontech company) and
PMD2.G (map is as shown in figure 11, is purchased from Clontech company) is transfected in 3:2:1 ratio into 6 orifice plates, after transfection 16h more
It uses the DMEM culture medium that serum content is 3% instead to continue to cultivate 48h, observes green fluorescence, illustrate the recombinant slow virus of packaging
It is secreted into extracellular culture medium, collects cells and supernatant at this time, it includes recombinant slow virus.
The transduction of 1.2 slow virus and the foundation of stable expression cell line
1.2.1 lentiviruses transduction is tested
The HEK-293T cell of culture is spread to 6 orifice plates, 3 × 105A cells/well reaches 70% left side to cell fusion degree
The right side, the 2mL recombinant slow virus that 1.1.3 is prepared access in 6 orifice plates, and after inoculation 48 hours, fluorescence microscopy under the microscope, is sent out
Now about 80% cell has fluorescence, it was demonstrated that lentiviruses transduction success, target gene anti-new castle disease virus NP protein nano antibody-
HRP (name are as follows: NDV-NP-Nb5-HRP) successfully passes lentiviruses transduction and enters in cell.
1.2.2 the foundation of stable expression cell line
A: the construction method of stable expression cell line
Above-mentioned lentivirus mediated is transfected into the cell training that the successfully HEK-293T cell with green fluorescence is passaged to 10cm
Support in ware, when density it is long to 90% or so when, then will sieve using the cell of selected by flow cytometry apoptosis expressing green fluorescent protein
The cell with green fluorescence of choosing, which is spread using limiting dilution assay to 96 porocyte culture plates, to be continued to screen, and finally filters out 1 plant
Growth conditions are good and have the cell strain of green fluorescence, carry out subsequent qualification test；The cell strain culture filtered out simultaneously
Cell conditioned medium use following preparations NDVNP albumen (LaSota plants of nucleocapsid proteins of 1.1 newcastle disease virus of such as embodiment 2
Expression and purification) be envelope antigen, the activity of HRP in the supernatant of cell line that Salmonella detection is surely sieved；Supernatant does difference
Doubling dilution after, indirect ELISA detects the NDV-NP-Nb5-HRP of steady sieve cell system secretion and the specific binding feelings of antigen
Condition.
B: interpretation of result
Fig. 2 is that the HEK-293 cell line for stablizing expression NDV-NP-Nb5-HRP of screening observes knot under fluorescence microscope
Fruit, the results show that the good cell strain of the production status filtered out is in fluorescence microscopy all equal fluoresced greens of cell under the microscope,
The cell strain of name screening is HEK-293T-NDV-Nb5-HRP；
Fig. 3 is the activity of HRP in the supernatant for the cell line that Salmonella detection is surely sieved, the results show that the cell line of screening
In the anti-new castle disease virus NP albumen Nb5-HRP fusion protein of secreting, expressing, Nb5 and HRP still have bioactivity, and Nb5 still has
There are the ability of the ability antigen binding in conjunction with NDV-NP proteantigen, and the ability of HRP and substrate reactions；
Fig. 4 is that indirect ELISA detects the NDV-NP-Nb5-HRP of steady sieve cell system secretion and the specific binding water of antigen
It is flat, the results show that the NDV-NP-Nb5-HRP of the cell line secretes of screening can be with the specific binding of antigen, and NDV-
NP-Nb5-HRP potency is 1:1000.
1.2.3 the expression for the cell line nano antibody that Western blotting identification is established
By the stable expression cell line HEK-293T-NDV-Nb5-HRP of screening, with 3 × 105A cells/well, paving to 6 holes
In cell plates, after culture 2 days, cell and its culture supernatant are collected, 10min is centrifuged using 3000g, carries out SDS-PAGE, and turn
After film, according to conventional operation, the Westernblotting detection of destination protein expression is carried out.Utilize the monoclonal antibody of anti-His label
For primary antibody, the IgG of the sheep anti mouse of HRP label is secondary antibody.
Fig. 5 is NDV-NP-Nb5- in the cell pyrolysis liquid and culture supernatant of Western blotting detection screening cell
HRP expression quantity as a result, itself the result shows that, at expected 72kDa, there is brown band, illustration purpose albumen NDV-Nb5-
HRP successful expression is simultaneously secreted into cells and supernatant.
Embodiment 2
1, the immunological identification of building cell line secretes expression NDV-Nb5-HRP
The expression and purification of 1.1 LaSota plants of nucleocapsid proteins of newcastle disease virus
1) recombinant prokaryotic expression vector of amplification and the building of newcastle disease virus LaSota plants of NP genes
It is with reference to sequence with the NDV LaSota pnca gene group sequence (gene order number is AF077761) announced on GenBank
Column, are designed, by Xi'an, Qing Ke bioengineering limited liability company is synthesized using 5.0 software of Primer, wherein
Upstream primer NDV-NP-F:CGCATATGAGCAGCGTGTTCGATGAAT；
Downstream primer NDV-NP-R:TACTCGAGGTAACCCCAGTCGGTATC.
(be purchased from: the attenuated vaccine of the easy nation NDV in Qingdao includes LaSota epidemic disease to newcastle disease virus LaSota strain vaccine virus liquid
Seedling strain) 150 μ L, extract viral RNA using TRIzol method, reverse transcription obtains cDNA, then as template, RT-PCR amplification
Newcastle disease virus NP gene is obtained, pcr amplification product is then subjected to agarose gel electrophoresis, obtains amplification NP gene；Wherein,
Reverse transcription is 20 μ L systems (following reagent is purchased from Dalian treasured Bioisystech Co., Ltd), as shown in table 1:
1 reverse transcription system of table (20 μ L)
Reverse transcription condition is 50 DEG C, 30min, then 85 DEG C, 5s, obtains cDNA；
PCR amplification system (all reagents of PCR amplification are purchased from Beijing Quanshijin Biotechnology Co., Ltd), such as 2 institute of table
Show,
2 PCR amplification system of table
Amplification condition is 95 DEG C, 10min；95 DEG C, 30s；55 DEG C, 30s；72 DEG C, 2min, totally 30 recycle；72 DEG C,
10min；12 DEG C of terminations, 4 DEG C of preservations.
The PCR product that amplification obtains NP gene is subjected to double digestion processing, plastic recovery kit with EcoR I and Xho I
(TransGen Biotech) recycling obtains NP gene, and pET-28a carrier carries out double digestion processing with EcoR I and Xho I, with
After the NP gene that recycling obtains is attached, it is transferred to DH-5 α and is evenly coated on the LB plate containing kalamycin resistance, screening sun
Property grain, recombinant plasmid pET-28a-NDV-NP double digestion, and be sequenced identification it is correct after be transferred to competence BL21 (DE3) and be used for
Expression.
Fig. 6 a is the amplification of anti-new castle disease virus LaSota plants of NP gene, successfully obtains expected 1457bp as the result is shown
The segment of size；Fig. 6 b is the digestion qualification result of the recombinant prokaryotic expression vector of building, obtains NP gene and load as the result is shown
The correct recombinant vector of body pET-28a.Wherein, in Fig. 6 a and Fig. 6 b: M is nucleic acid Marker；1 and 2 be the NP gene of amplification；
The 3 NP genes obtained for digestion recombinant vector；4 carrier segments obtained for digestion recombinant vector.
2) expression and purification of newcastle disease virus LaSota plants of nucleocapsid proteins
The e. coli bl21 (DE3) that positive plasmid is converted when 37 DEG C of shake cultures to bacterium solution OD600 value are up to 0.6, is used
37 DEG C of induction 5h of final concentration of 1.0mM/L, IPTG take 15mL bacterium solution, and 6000g is centrifuged 10min, removes supernatant, are added and combine buffering
Precipitating is resuspended in liquid, and after sonicated, 6000g is centrifugated supernatant and deposit sample, and SDS-PAGE detection, testing result is such as
Shown in Fig. 7 a, determine that recombinant nucleocapsid protein is solubility expression.Utilize the positive chicken serum of anti-new castle disease virus, Western
Blotting detection, result is as shown in Figure 7b, and the NP albumen of prokaryotic expression has antigenicity.By the soluble NP egg of expression
It is white, illustrate to carry out protein purification referring to QIAGEN purification kit, purification of samples is detected through SDS-PAGE, as the result is shown albumen
It is purified well.M is albumen Marker in Fig. 7 a and Fig. 7 b；Lane 1 is empty carrier pET-28a control；Lane 2 is to lure
Lead expression cellular lysate liquid；Lane 3 is inclusion body；Lane 4 is supernatant after ultrasound；Lane 5 is the destination protein of purifying.
The reactivity detection of NDV-Nb5-HRP and the NDV-NP albumen of 1.2 secreting, expressings
The NDV-NP albumen of Prokaryotic expression, purification is coated with elisa plate, package amount 200ng/well, 4 DEG C of overnight incubations
Afterwards, 200 μ L confining liquid (PBST of 2.5% skimmed milk power), after 37 DEG C are closed 1 hour, cleaning solution PBST board-washing 3 times directly adds
Enter the cells and supernatant (comprising NDV-Nb5-HRP) of different dilutions (1:10-1:10000), after 37 DEG C are incubated for 1 hour, directly
It connects and TMB colour developing, the automatic microplate reader OD of ELISA is added45nmReading.
As shown in figure 3, the cell line HEK-293T-NDV-Nb5-HRP constructed as the result is shown, the nano antibody of secreting, expressing
NDV-Nb5-HRP still keeps ability of the nano antibody Nb5 in conjunction with NDV-NP proteantigen, and the HRP and substrate that express anti-
Answer ability (amino acid sequence of NP protein nano antibody as shown in SEQ ID NO:3, the amino acid sequence of HRP such as SEQ ID
Shown in NO:4).
Embodiment 3
Anti- NDV positive chicken serum blocks the antigen binding of NDV-Nb5-HRP and NDV-NP albumen
The NDV-NP albumen of prokaryotic expression is coated with elisa plate, the hole 100ng/, 4 DEG C are incubated overnight, 200 μ L confining liquids,
After 37 DEG C are closed 1 hour, the serum (1:10,1:20,1:40 and 1:80) of different dilutions is added in the hole ELISA, 37 DEG C
After being incubated for 1 hour, the diluted cells and supernatant of 1:1000 (including NDV-Nb5-HRP) is added in every hole, and 37 DEG C are incubated for 1 hour
Afterwards, it is directly added into TMB colour developing after ten minutes, 3mol H is added2SO4Color development stopping, the automatic microplate reader OD of ELISA45nmReading.
Fig. 8 a and b are that the combination of NDV-NP-Nb5-HRP and NDV-NP albumen in newcastle disease virus antibody chicken serum is horizontal,
Itself the result shows that, the middle OD of positive chicken serum culture450nmValue reduces, and illustrates that the positive chicken serum of anti-new castle disease virus can be fine
Blocking NDV-Nb5-HRP and NDV-NP albumen react, and OD in negative serum450nmValue does not reduce, and illustrates anti-Newcastle Disease
The negative chicken serum of poison cannot block reacting for NDV-Nb5-HRP and NDV-NP albumen.
In summary, the fusion of anti-new castle disease virus nucleocapsid protein Nb5-HRP fusion protein is carried by building
Slow virus carrier, and transfect into HEK-293T cell, constitute stablize expression anti-new castle disease virus nucleocapsid protein Nb5-
The cell line of HRP fusion protein；Include NDV-Nb5-HRP in supernatant of the constructed cell line through cultivating, can directly pass through
The supernatant is directly added into positive chicken serum, and for detecting whether chicken infects newcastle disease virus, serum is examined compared with prior art
Survey technology avoids the secondary antibody (indirect ELISA) for preparing the anti-chicken of enzyme label or the monoclonal antibody for needing anti-NDV nucleocapsid protein
Situation, simplifies detection process, and save the cost, the efficiency of detection can be improved, and has application well in serum detection technique
Prospect.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and shown here as with attached drawing.
Sequence table
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>cell line of expression anti-new castle disease virus fusion protein and its construction method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 461
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ctgcaggaga cagtacatat gaaataccta ttgcctacgg cagccgctgg attgttatta 60
ctcgcggccc agccggccat ggcccaggtg cagctgcagg agtctggggg aggctcggtg 120
caggctggag ggtctctgag actctcctgt gcagcctctg gatacaagtt tagtcccatg 180
tgtatgggct ggttccgcca ggctccaggg aaggagcgcg agggggtcgc agcgcttgat 240
agtgatggtc tgacaagcta cgcagactcc gtgaagggcc gattcaccat ctccaaagac 300
aacgccaaga acactctcta tctgcaaatg aacagcctga cacctgagga cactgccatg 360
tactactgtg cggcgagacg tagccggtac tgctctcgag tacctgagat ctatggctac 420
tggggccagg ggacccaggt caccgtctcc tcagcggccg c 461
<210> 2
<211> 933
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgcagctga cccccacatt ctacgataac tcttgtccca acgtgtccaa catcgttcgt 60
gacatcatcg tgaatgagct gaggagcgac cctaggatcg ccgccagcat tctgaggctg 120
cacttccacc tggataacac caccagcttt cgtaccgaaa aggacgcttt cggcaacgcc 180
aacagcgctc gtggcttcag cgtgatcgat cgtatgaaag ccgccgtgga aagcgcttgt 240
cccggcacag tgtgactgct tcgtgaatgg ctgcgacgcc tccattttac ttgtgccgat 300
ttattaacca ttgccgccca gcagtccgtg acactggccg gcggacctag ctggagagtg 360
cctctgggtc gtagggattc tttacaagcc tttttagatt tagccaacgc caatttaccc 420
gcccctttct tcactttacc tcagctcaag gacagcttca gaaacgtggg tttaaatagg 480
agcagcgatt tagtggcttt atccggcggc cacacatttg gcaagagcca gtgtcgtttc 540
attatggatc gtctgtacaa tttcagcaac accggtttac ccgaccccac actgaacacc 600
acctatctcc agactttaag aggtttatgc cctttaaacg gcaatctgtc cgctttagtg 660
gatttcgact tacgtacccc cacaatcttc gacaacaaat actacgtgaa tttagaggag 720
cagaagggtt taatccagag cgaccaagaa ctgttttcca gccccgatgc caccgacacc 780
atccctctgg tgaggagctt cgccaactcc acccagacct tcttcaacgc ctttgtggag 840
gccatggatc gtatgggcaa cattacccct ctgaccggca cccaaggtca gattcgtagg 900
aactgtaggg tggtgaatag caacagcgat tta 933
<210> 3
<211> 122
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Ala Ala Ser Gly Tyr Lys Phe Ser Pro Met Cys Met Gly
20 25 30
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu
35 40 45
Asp Ser Asp Gly Leu Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Thr Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Arg
85 90 95
Ser Arg Tyr Cys Ser Arg Val Pro Glu Ile Tyr Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120
<210> 4
<211> 311
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser
1 5 10 15
Asn Ile Val Arg Asp Ile Ile Val Ile Leu Arg Leu His Phe His Asp
20 25 30
Cys Phe Val Asn Gly Cys Asp Ala Ser Ile Leu Leu Asp Asn Thr Thr
35 40 45
Ser Phe Asn Glu Leu Arg Ser Asp Pro Arg Ile Ala Ala Ser Arg Thr
50 55 60
Glu Lys Asp Ala Phe Gly Asn Ala Asn Ser Ala Arg Gly Phe Ser Val
65 70 75 80
Ile Asp Arg Met Lys Ala Ala Val Glu Ser Ala Cys Pro Gly Thr Val
85 90 95
Ser Cys Ala Asp Leu Leu Thr Ile Ala Ala Gln Gln Ser Val Thr Leu
100 105 110
Ala Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu
115 120 125
Gln Ala Phe Leu Asp Leu Ala Asn Ala Asn Leu Pro Ala Pro Phe Phe
130 135 140
Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg
145 150 155 160
Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Ser
165 170 175
Gln Cys Arg Phe Ile Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly
180 185 190
Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Asp Leu Arg Thr Pro
195 200 205
Thr Ile Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu Gln Lys Gly
210 215 220
Leu Ile Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asp Ala Thr Asp
225 230 235 240
Thr Ile Pro Leu Val Arg Ser Phe Ala Asn Ser Thr Gln Thr Phe Phe
245 250 255
Asn Ala Phe Val Glu Gln Thr Leu Arg Gly Leu Cys Pro Leu Asn Gly
260 265 270
Asn Leu Ser Ala Leu Val Asp Phe Ala Met Asp Arg Met Gly Asn Ile
275 280 285
Thr Pro Leu Thr Gly Thr Gln Gly Gln Ile Arg Arg Asn Cys Arg Val
290 295 300
Val Asn Ser Asn Ser Asp Leu
305 310
Claims (8)
1. a kind of cell line for expressing anti-new castle disease virus NP protein nano antibody-HRP fusion protein, which is characterized in that described
Cell line is C201981 in the deposit number of China typical culture collection center；Depositary institution address: Hubei Wuhan, Wuhan are big
It learns.
2. a kind of cell of expression anti-new castle disease virus NP protein nano antibody-HRP fusion protein as described in claim 1
The construction method of system, which comprises the following steps:
A: Newcastle disease virus NP albumen will composite coding anti-new castle disease virus NP protein nano antibody and HRP fusion: be encoded
The nucleotide sequence of nano antibody and HRP directly merge, and the flexible sequence connection in centre synthesizes recombinant fusion gene；
B: the recombined lentivirus vector for expressing the recombinant fusion gene is constructed: by the recombinant fusion gene through digestion
Afterwards, it is connected on slow virus carrier, obtains recombined lentivirus vector；Wherein, slow virus carrier PLVX-IRES-ZsGreen1；
C: the packaging of recombined lentivirus vector: the recombined lentivirus vector and 2 helper plasmids are transfected to the HEK- of culture
In 293T cell, secondary culture, screening obtains the cell of expression anti-new castle disease virus NP protein nano antibody and HRP fusion protein
System.
3. expressing the cell line of anti-new castle disease virus NP protein nano antibody-HRP fusion protein as claimed in claim 2
Construction method, which is characterized in that the nucleotide sequence of the Newcastle disease virus NP protein nano antibody such as SEQ ID NO:1 institute
Show, the nucleotide sequence of the HRP is as shown in SEQ ID NO:2.
4. the cell line of expression anti-new castle disease virus NP protein nano antibody-HRP fusion protein as claimed in claim 2,
It is characterized in that, the helper plasmid is psPAX2 and pMD2.G.
5. a kind of cell line for expressing anti-new castle disease virus NP protein nano antibody-HRP fusion protein is by claim 2-4
The building of construction method described in any one.
6. a kind of anti-new castle disease virus NP protein nano antibody-HRP fusion protein is by the cell line secretes of claim 1 or 5
Expression.
7. a kind of anti-new castle disease virus NP protein nano antibody-HRP fusion protein as claimed in claim 6 is in chicken serum
Application in anti-new castle disease virus antibody test.
8. a kind of anti-new castle disease virus NP protein nano antibody-HRP fusion protein as claimed in claim 6 is anti-in newcastle disease
Application in the detection of body positive chicken serum.
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| CN112852746A (en) * | 2021-02-04 | 2021-05-28 | 中吉智药(南京)生物技术有限公司 | Large-scale lentivirus gene medicine preparation system and method based on Cre recombinase induction |
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| CN111551744A (en) * | 2020-05-15 | 2020-08-18 | 安徽中起生物科技有限公司 | Newcastle disease virus N protein IgY antibody colloidal carbon detection test paper and application thereof |
| CN112852746A (en) * | 2021-02-04 | 2021-05-28 | 中吉智药(南京)生物技术有限公司 | Large-scale lentivirus gene medicine preparation system and method based on Cre recombinase induction |
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